Methods of optimizing antibody variable region binding affinity

ABSTRACT

The invention provides a method of conferring donor CDR binding affinity onto an antibody acceptor variable region framework. The invention also provides a method of simultaneously grafting and optimizing the binding affinity of a variable region binding fragment. A method of optimizing the binding affinity of an antibody variable region is also provided.

BACKGROUND OF THE INVENTION

[0001] This invention relates generally to a method of monoclonalantibody production and specifically to the simultaneous in vitroaffinity optimization of multiple distinct domains of a variable regionof a monoclonal antibody.

[0002] The War on Cancer is entering its third decade and recent yearshave shown tremendous progress in the understanding of cancerdevelopment and progression yet there has been only marginal decreasesin death rates from most types of cancer. Standard chemotherapy andradiation therapy generally involve treatment with therapeutic agentsthat impact not only cancer cells but other highly proliferative cellsof the body, often leading to debilitating side effects. Thus, it isdesirable to identify therapeutic agents with a higher degree ofspecificity for the carcinogenic lesion.

[0003] The discovery of monoclonal antibodies (mAbs) in the 1970'sprovided great hope for the reality of creating therapeutic moleculeswith high specificity. Antibodies that bind to tumor antigens wouldprovide specific targeting agents for cancer therapy. However, while thedevelopment of monoclonal antibodies has provided a valuable diagnosticreagent, certain limitations restrict their use as therapeutic entities.

[0004] A limitation encountered when attempts are made to use mAbs astherapeutic agents is that since mAbs are developed in non-humanspecies, usually mouse, they elicit an immune response in humanpatients. Chimeric antibodies join the variable region of the non-humanspecies, which confers binding activity, to a human constant region.However, the chimeric antibody is often still immunogenic and it istherefore necessary to further modify the variable region.

[0005] One modification is the grafting of complementarity-determiningregions, (CDRs) which are in part antigen binding onto a human antibodyvariable framework. However, this approach is imperfect because CDRgrafting often diminishes the binding activity of the resultinghumanized mAb. Attempts to regain binding activity require laborious,step-wise procedures which have been pursued essentially by a trial anderror type of approach. For example, one difficulty in regaining bindingaffinity is because it is difficult to predict which framework residuesserve a critical role in maintaining antigen binding affinity andspecificity. Consequently, while antibody humanization methods that relyon structural and homology data are used, the complexity that arisesfrom the large number of framework residues potentially involved inbinding activity has prevented success.

[0006] Combinatorial methods have been applied to restore bindingaffinity, however, these methods require sequential rounds ofmutagenesis and affinity selection that can both be laborious andunpredictable.

[0007] Thus, there exists a need for efficient and reliable methods forproducing human monoclonal antibodies which exhibit comparable orenhanced binding affinities to their non-human counterparts. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0008] The invention provides a method of conferring donor CDR bindingaffinity onto an antibody acceptor variable region framework. The methodconsists of: (a) constructing a population of altered antibody variableregion encoding nucleic acids, said population comprising encodingnucleic acids for an acceptor variable region framework containing aplurality of different amino acids at one or more acceptor frameworkregion amino acid positions and donor CDRs containing a plurality ofdifferent amino acids at one or more donor CDR amino acid positions; (b)expressing said population of altered variable region encoding nucleicacids, and (c) identifying one or more altered variable regions havingbinding affinity substantially the same or greater than the donor CDRvariable region. The acceptor variable region framework can be a heavyor light chain variable region framework and the populations of heavyand light chain altered variable regions can be expressed alone toidentify heavy or light chains having binding affinity substantially thesame or greater than the donor CDR variable region. The populations ofheavy and light chains additionally can be coexpressed to identifyheteromeric altered variable region binding fragments. The inventionalso provides a method of simultaneously grafting and optimizing thebinding affinity of a variable region binding fragment. The methodconsists of: (a) constructing a population of altered heavy chainvariable region encoding nucleic acids comprising an acceptor variableregion framework containing donor CDRs and a plurality of differentamino acids at one or more framework region and CDR amino acidpositions; (b) constructing a population of altered light chain variableregion encoding nucleic acids comprising an acceptor variable regionframework containing donor CDRs and a plurality of different amino acidsat one or more framework regions and CDR amino acid positions; (c)coexpressing said populations of heavy and light chain variable regionencoding nucleic acids to produce diverse combinations of heteromericvariable region binding fragments, and (d) identifying one or moreheteromeric variable region binding fragments having affinitysubstantially the same or greater than the donor CDR heteromericvariable region binding fragment. A method of optimizing the bindingaffinity of an antibody variable region is also provided. The methodconsists of: (a) constructing a population of antibody variable regionencoding nucleic acids, said population comprising two or more CDRscontaining a plurality of different amino acids at one or more CDR aminoacid positions; (b) expressing said population of variable regionencoding nucleic acids, and (c) identifying one or more variable regionshaving binding affinity substantially the same or greater than the donorCDR variable region. The variable region populations can be heavy orlight chains and can be expressed as individual populations or they canbe coexpressed to produce heteromeric variable region binding fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 shows the alignment of anti-CD40 variable region and humantemplate amino acid sequences.

[0010]FIG. 2 shows binding reactivity of humanized anti-CD40 variants.

[0011]FIG. 3 shows molecular modeling of anti-CD40 variant CW43.

[0012]FIG. 4 shows a comparison of the quantitation of murine frameworkresidues in active variants from two libraries.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The invention is directed to a method of conferring donor CDRbinding affinity onto an antibody acceptor variable region framework.The method effectively combines CDR grafting procedures and affinityreacquisition of the grafted variable region into a single step. Themethods of the invention also are applicable for affinity maturation ofan antibody variable region. The affinity maturation process can besubstituted for, or combined with the affinity reacquisition functionwhen being performed during a CDR grafting procedure. Alternatively, theaffinity maturation procedure can be performed independently from CDRgrafting procedures to optimize the binding affinity of variable region,or an antibody. An advantage of combining grafting and affinityreacquisition procedures, or affinity maturation, is the avoidance oftime consuming, step-wise procedures to generate a grafted variableregion, or antibody, which retains sufficient binding affinity fortherapeutic utility. Therefore, therapeutic antibodies can be generatedrapidly and efficiently using the methods of the invention. Suchadvantages beneficially increase the availability and choice of usefultherapeutics for human diseases as well as decrease the cost to thedeveloper and ultimately to the consumer.

[0014] In one embodiment, the invention is directed to methods ofproducing grafted heavy and light chain variable regions having similaror better binding affinity as the CDR donor variable region. Whencoexpressed, the grafted heavy and light chain variable regions assembleinto variable region binding fragments having similar or better bindingaffinity as the donor antibody or variable region binding fragmentsthereof. The grafting is accomplished by generating a diverse library ofCDR grafted variable region fragments and then screening the library forbinding activity similar or better than the binding activity of thedonor. A diverse library is generated by selecting acceptor frameworkpositions that differ at the corresponding position compared to thedonor framework and making a library population containing of allpossible amino acid residue changes at each of those positions togetherwith all possible amino acid residue changes at each position within theCDRs of the variable region. The grafting is accomplished by splicing apopulation of encoding nucleic acids for the donor CDR containingspecies representing all possible amino acid residues at each CDRposition into a population of encoding nucleic acids for an antibodyacceptor variable region framework which contains species representingall possible amino acid residue changes at the selected frameworkpositions. The resultant population encodes the authentic donor andacceptor framework amino acid sequences as well as all possiblecombinations and permutations of these sequences with each of the 20naturally occurring amino acids at the changed positions.

[0015] In another embodiment, the invention is directed to methods ofproducing grafted heavy and light chain variable regions, andheteromeric binding fragments thereof, having similar or better bindingaffinity as the CDR donor variable region. As described above, thegrafting is accomplished by generating a diverse library of CDR graftedvariable region fragments and then screening the library for bindingactivity similar or better than the binding activity of the donor.However, the diverse library is generated by selecting acceptorframework positions that are predicted to affect CDR binding affinityand making a library population containing of all possible amino acidresidue changes at each of those positions or subsets of the selectedamino acid positions together with all possible amino acid residuechanges at each position within the CDRs of the variable region, orsubsets of CDR positions. The grafting is accomplished by splicing apopulation of encoding nucleic acids for the donor CDR containing theselected position changes into a population of encoding nucleic acidsfor an antibody acceptor variable region framework which contains theselected position changes.

[0016] In yet another embodiment, the invention is directed to theoptimization of binding affinity of an antibody variable region. Theoptimization is accomplished by generating a library of variable regionswhich contain all possible amino acid residue changes at each amino acidposition within two or more CDRS. When expressed and screened forbinding activity, the variable region, or heavy and light chainheteromeric binding fragments, those species within the population areselected that contain increased or decreased binding activity comparedto the parent molecule as optimal binders. Libraries containing subsets,representing less than all amino acid positions within the CDRs, cansimilarly be generated and screened for selecting optimal bindingvariable regions and heteromeric binding fragments thereof.

[0017] As used herein, the term “CDR” or “complementarity determiningregion” is intended to mean the non-contiguous antigen combining sitesfound within the variable region of both heavy and light chainpolypeptides. These particular regions have been described by Kabat etal., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences ofprotein of immunological interest. (1991), and by Chothia et al., J.Mol. Biol. 196:901-917 (1987) and by MacCallum et al., J. Mol. Biol.262:732-745 (1996) where the definitions include overlapping or subsetsof amino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orgrafted antibodies or variants thereof is intended to be within thescope of the term as defined and used herein. The amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 1 as a comparison. TABLE 1 CDRDefinitions Kabat¹ Chothia² MacCallum³ V_(H) CDR1 31-35 26-32 30-35V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3 95-102 96-101 93-101 V_(L) CDR124-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L) CDR3 89-97 91-9689-96

[0018] As used herein, the term “framework” when used in reference to anantibody variable region is entered to mean all amino acid residuesoutside the CDR regions within the variable region of an antibody.Therefore, a variable region framework is between about 100-120 aminoacids in length but is intended to reference only those amino acidsoutside of the CDRs.

[0019] As used herein, the term “framework region” is intended to meaneach domain of the framework that is separated by the CDRs. Therefore,for the specific example of a heavy chain variable region and for theCDRs as defined by Kabat et al., framework region 1 corresponds to thedomain of the variable region encompassing amino acids 1-30; region 2corresponds to the domain of the variable region encompassing aminoacids 36-49; region 3 corresponds to the domain of the variable regionencompassing amino acids 66-94, and region 4 corresponds to the domainof the variable region from amino acids 103 to the end of the variableregion. The framework regions for the light chain are similarlyseparated by each of the light claim variable region CDRs. Similarly,using the definition of CDRs by Chothia et al. or McCallum et al. theframework region boundaries are separated by the respective CDR terminias described above.

[0020] As used herein,the term “donor” is intended to mean a parentantibody molecule or fragment thereof from which a portion is derivedfrom, given or contributes to another antibody molecule or fragmentthereof so as to confer either a structural or functional characteristicof the parent molecule onto the receiving molecule. For the specificexample of CDR grafting, the parent molecule from which the grafted CDRsare derived is a donor molecule. The donor CDRs confer binding affinityof the parent molecule onto the receiving molecule. It should beunderstood that a donor molecule does not have to be from a differentspecies as the receiving molecule of fragment thereof. Instead, it issufficient that the donor is a separate and distinct molecule.

[0021] As used herein, the term “acceptor” is intended to mean anantibody molecule or fragment thereof which is to receive the donatedportion from the parent or donor antibody molecule or fragment thereof.An acceptor antibody molecule or fragment thereof is therefore impartedwith the structural or functional characteristic of the donated portionof the parent molecule. For the specific example of CDR grafting, thereceiving molecule for which the CDRs are grafted is an acceptormolecule. The acceptor antibody molecule or fragment is imparted withthe binding affinity of the donor CDRs or parent molecule. As with adonor molecule, it is understood that an acceptor molecule does not haveto be from a different species as the donor.

[0022] A “variable region” when used in reference to an antibody or aheavy or light chain thereof is intended to mean the amino terminalportion of an antibody which confers antigen binding onto the moleculeand which is not the constant region. The term is intended to includefunctional fragments thereof which maintain some of all of the bindingfunction of the whole variable region. Therefore, the term “heteromericvariable region binding fragments” is intended to mean at least oneheavy chain variable region and at least one light chain variableregions or functional fragments thereof assembled into a heteromericcomplex. Heteromeric variable region binding fragments include, forexample, functional fragments such as Fab, F(ab)₂, Fv, single chain Fv(scfv) and the like. Such functional fragments are well known to thoseskilled in the art. Accordingly, the use of these terms in describingfunctional fragments of a heteromeric variable region is intended tocorrespond to the definitions well known to those skilled in the art.Such terms are described in, for example, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1989);Molec. Biology and Biotechnoloqy: A Comprehensive Desk Reference (Myers,R. A. (ed.), New York: VCH Publisher, Inc.); Huston et al., CellBiophysics, 22:189-224 (1993); Plückthun and Skerra, Meth. Enzymol.,178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, SecondEd., Wiley-Liss, Inc., New York, N.Y. (1990).

[0023] As used herein, the term “population” is intended to refer to agroup of two or more different molecules. A population can be as largeas the number of individual molecules currently available to the user orable to be made by one skilled in the art. Populations can be as smallas 2-4 molecules or as large as 10¹³ molecules. An example where a smallpopulation can be useful is where one wishes to optimize bindingaffinity of a variable region or of heteromeric binding fragments bycompiling beneficial differences from a small number of parent moleculeshaving similar binding affinity into a single variable binding fragmentspecies. An example of where large populations, including as large as10⁸ or greater different molecules, can be desired is where all possiblecombinations of amino acids differences between donor and acceptor atall positions within a variable region are to be generated in order toobtain maximum diversity and increase the efficiency of compilingbeneficial changes. In some embodiments, populations are between about 5and 10 different species as well as up to hundreds or thousands ofdifferent species. The populations can be diverse or redundant dependingon the intent and needs of the user. Those skilled in the art will knowwhat size and diversity of a population is suitable for a particularapplication.

[0024] As used herein, the term “altered” when used in reference to anantibody variable region is intended to mean a heavy or light chainvariable region that contains one or more amino acid changes in aframework region, a CDR or both compared to the parent amino acidsequence at the changed position. Where an altered variable region isderived from or composed of different donor and acceptor regions, thechanged amino acid residues within the altered species are to becompared to their respective amino acid positions within the parentdonor and acceptor regions. For example, a variable region containingdonor CDRs grafted into an acceptor framework and containing one or moreamino acid changes within the framework regions and one or more aminoacid changes within the CDRs will have amino acids residues at thechanged framework region positions different than the residues at thecomparable positions in the acceptor framework. Similarly, such analtered variable region will have amino acid residues at the changed CDRpositions different than the residues at the comparable positions in thedonor CDRs.

[0025] As used herein, the term “nucleic acid” or “nucleic acids” isintended to mean a single- or double-stranded DNA or RNA molecule. Anucleic acid molecule of the invention can be of linear, circular orbranched configuration, and can represent either the sense or antisensestrand, or both, of a native nucleic acid molecule. The term also isintended to include nucleic acid molecules of both synthetic and naturalorigin. A nucleic acid molecule of natural origin can be derived fromany animal, such as a human, non-human primate, mouse, rat, rabbit,bovine, porcine, ovine, canine, feline, or amphibian, or from a lowereukaryote, such as Drosophila, C. elegans or yeast. A synthetic nucleicacid includes, for example, chemical and enzymatic synthesis. The term“nucleic acid” or “nucleic acids” is similarly intended to includeanalogues of natural nucleotides which have similar functionalproperties as the referenced nucleic acid and which can be utilized in amanner similar to naturally occurring nucleotides and nucleosides.

[0026] As used herein, the term “coexpressing” is intended to mean theexpression of two or more molecules by the same cell. The coexpressedmolecules can be polypeptides or encoding nucleic acids. Thecoexpression can be, for example, constitutive or inducible. Suchnucleic acid sequences can also be expressed simultaneously or,alternatively, regulated independently. Various combinations of thesemodes of coexpression can additionally be used depending on the numberand intended use of the variable region encoding nucleic acids. The termis intended to include the coexpression of members originating fromdifferent populations in the same cell. For example, populations ofmolecules can be coexpressed where single or multiple different speciesfrom two or more populations are expressed in the same cell. A specificexample includes the coexpression of heavy and light chain variableregion populations where at least one member from each population isexpressed together in the same cell to produce a library of cellscoexpressing different species of heteromers variable region bindingfragments. Populations which can be coexpressed can be as small as 2different species within each population. Additionally, the number ofmolecules coexpressed from different populations also can be as large as10⁸ or greater, such as in the case where multiple amino acid positionchanges of multiple framework regions or CDRs in both heavy and lightchain antibody variable region populations are produced and coexpressed.Numerous different sized populations of encoding nucleic acids inbetweenthe the above ranges and greater can also be coexpressed. Those skilledin the art know, or can determine, what modes of coexpression can beused to achieve a particular goal or satisfy a desired need.

[0027] As used herein, the term “identifying” is intended to meandetecting by a qualitative or quantitative means, a variable region oraltered variable of the invention by functional or biochemicalproperties, including, for example, binding affinity of catalyticactivity.

[0028] As used herein the term “binding affinity” is intended to meanthe strength of a binding interaction and therefore includes both theactual binding affinity as well as the apparent binding affinity. Theactual binding affinity is a ratio of the association rate over thedisassociation rate. Therefore, conferring or optimizing bindingaffinity includes altering either or both of these components to achievethe desired level of binding affinity. The apparent affinity caninclude, for example, the avidity of the interaction. For example, abivalent heteromeric variable region binding fragment can exhibitaltered or optimized binding affinity due to its valency.

[0029] As used herein, the term “optimizing” when used in reference to avariable region or a functional fragment thereof is intended to meanthat the binding affinity of the variable region has been modifiedcompared to the binding affinity of a parent variable region or a donorvariable region. A variable region exhibiting optimized activity canexhibit, for example, higher affinity or lower affinity binding, orincreased or decreased association or dissociation rates compared to anunaltered variable region. A variable region exhibiting optimizedactivity also can exhibit increased stability such as increasedhalf-life in a particular organism. For example, an antibody activitycan be optimized to increase stability by decreasing susceptibility toproteolysis. An antibody exhibiting optimized activity also can exhibitlower affinity binding, including decreased association rates orincreased dissociation rates, if desired. An optimized variable regionexhibiting lower affinity binding is useful, for example, forpenetrating a solid tumor. In contrast to a higher affinity variableregion, which would bind to the peripheral regions of the tumor butwould be unable to penetrate to the inner regions of the tumor due toits high affinity, a lower affinity variable region would beadvantageous for penetrating the inner regions of the tumor. As withoptimization of binding affinities above, optimization of a catalyticvariable region can be, for example, increased or decreased catalyticrates, disassociation constants or association constants.

[0030] As used herein, the term “substantially the same” when used inreference to binding affinity is intended to mean similar or identicalbinding affinities where one molecule has a binding affinity constantthat is similar to another molecule within the experimental variabilityof the affinity measurement. The experimental variability of the bindingaffinity measurement is dependent upon the specific assay used and isknown to those skilled in the art.

[0031] The invention provides a method for conferring donor CDR bindingaffinity onto an antibody acceptor variable region framework. The methodconsists of: (a) constructing a population of altered antibody variableregion encoding nucleic acids, the population consisting of encodingnucleic acids for an acceptor variable region framework containing aplurality of different amino acids at one or more acceptor frameworkregion amino acid positions and donor CDRs containing a plurality ofdifferent amino acids at one or more donor CDR amino acid positions; (b)expressing the population of altered variable region encoding nucleicacids, and (c) identifying one or more altered variable regions havingbinding affinity substantially the same or greater than the donor CDRvariable region.

[0032] The process of producing human antibody forms from nonhumanspecies involves recombinantly splicing CDRs from a nonhuman donorantibody into a human acceptor framework region to confer bindingactivity onto the resultant grafted antibody, or variable region bindingfragment thereof. The process of grafting, referred to as the procedurefor splicing CDRs into a framework, while mechanically simple it almostalways results in a grafted antibody that exhibits a substantial loss inbinding affinity. Although donor and acceptor variable regions arestructurally similar, the process nevertheless combines CDR bindingdomains with a heterologous acceptor region, resulting in aconformationally imperfect setting for the binding residues of thegrafted antibody. Therefore, once the CDR-grafted antibody, or variableregion binding fragment is made, it requires subsequent rounds ofmolecular engineering to reacquire binding affinity comparable to thedonor antibody. The present invention combines these steps such that CDRgrafting and binding reacquisition occur in a single simultaneousprocedure. The method is also applicable to optimizing the bindingaffinity of an antibody, or variable region binding fragmentsimultaneous with CDR grafting and to optimizing an antibody or variableregion binding fragment in a single procedure without including the CDRgrafting process.

[0033] The methods of the invention confer or impart donor CDR bindingaffinity onto an antibody acceptor variable region framework in aprocedure which achieves grafting of donor CDRs and affinityreacquisition in a simultaneous process. The methods similarly can beused, either alone or in combination with CDR grafting, to modify oroptimize the binding affinity of a variable region. The methods forconferring donor CDR binding affinity onto an acceptor variable regionare applicable to both heavy and light chain variable regions and assuch can be used to simultaneous graft and optimize the binding affinityof an antibody variable region.

[0034] The methods for conferring donor CDR binding affinity onto avariable region involve identifying the relevant amino acid positions inthe acceptor framework that are known or predicted to influence a CDRconformation, or that are known or predicted to influence the spacialcontext of amino acid side chains within the CDR that participate inbinding, and then generate a population of altered variable regionspecies that incorporate a plurality of different amino acid residues atthose positions. For example, the different amino acid residues at thosepositions can be incorporated either randomly or with a predeterminedbias and can include all of the twenty naturally occurring amino acidresidues at each of the relevant positions. Subsets, including less thanall of the naturally occurring amino acids can additionally be chosenfor incorporation at the relevant framework positions. Including aplurality of different amino acid residues at each of the relevantframework positions ensures that there will be at least one specieswithin the population that will have framework changes which allows theCDRs to reacquire their donor binding affinity in the context of theacceptor framework variable region.

[0035] In addition to the framework changes at selected amino acidpositions, the CDRs also are altered to contain a plurality of differentamino acid residue changes at all or selected positions within the donorCDRS. For example, random or biased incorporation of the twentynaturally occurring amino acid residues, or preselected subsets, arealso introduced into the donor CDRs to produce a diverse population ofCDR species. Including a diverse population of different CDR variantspecies ensures that beneficial changes in the framework positions arenot neutralized by a conformationally incompatible residue in a donorCDR. Inclusion of CDR variant species into the diverse population ofvariable regions also allows for the generation of variant species thatexhibit optimized binding affinity for a predetermined antigen.

[0036] The resultant population of CDR grafted variable regionsdescribed above will therefore contain, at the relevant frameworkpositions and at the selected CDR positions, a species corresponding tothe authentic parent amino acid residue at each position as well as adiverse number of different species which correspond to the possiblecombinations and permutations of the authentic parent amino acidresidues together with the variant residues at each of the relevantframework and selected CDR positions. Such a diverse population of CDRgrafted variable regions are screened for an altered variable regionspecies which retains donor CDR binding activity, or optimized bindingactivity.

[0037] One advantage of the methods of the invention is that they do notlimit the choice of acceptor variable regions applicable, or expected tobe successful, for receiving CDRs from the donor molecule. For example,when choosing an acceptor region it can be desirable, or in somecircumstances even required, to select an acceptor that is closelysimilar to the variable region amino acid sequence harboring the donorCDRs because the CDR conformation in the grafted variable region willlikely be more similar to that of the donor. However, selecting similarframework region sequences between the donor and acceptor variableregions still does not provide which residues, out of the differences,actually play a role in CDR binding affinity of the grafted variableregion. Selection of similar acceptor frameworks therefore only limitsthe number of possible residues which to investigate in order toreacquire binding affinity onto the grafted variable region. The methodsof the invention circumvent this problem by producing a library of allpossible or relevant changes in the acceptor framework, and thenscreening those variable regions, or heteromeric binding fragmentsthereof for species that maintain or exhibit increased binging affinitycompared to the donor molecule. Therefore, the applicability is notpreconditioned on the availability or search for an acceptor frameworkvariable region similar to that of the donor.

[0038] Selection of the relevant framework amino acid positions to altercan depend on a variety of criteria well known to those skilled it theart. As described above, one criteria for selecting relevant frameworkamino acids to change can be the relative differences in amino acidframework residues between the donor and acceptor molecules. Selectionof relevant framework positions to alter using this approach is simpleand has the advantage of avoiding any subjective bias in residuedetermination or any inherent bias in CDR binding affinity contributionby the residue. Criteria other than relatedness of amino acid residuescan be used for selecting relevant framework positions to alter. Suchcriteria can be used in combination with, or alternative to theselection of framework positions having divergent amino acid residues.These additional criteria are described further and similarly are wellknown to those skilled in the art.

[0039] Another criteria which can be used for determining the relevantamino acid positions to change can be, for example, selection offramework residues that are known to be important, or contribute to CDRconformation. For example, canonical framework residues play such a rolein CDR conformation or structure. Such residues can be considered to berelevant to change for a variety of reasons, including for example,their new context of being associated with heterologous CDR sequences inthe grafted variable region. Targeting of a canonical framework residueas a relevant position to change can identify a more compatible aminoacid residue in context with its associated donor CDR sequence.Additionally, targeting of canonical residues can allow for theidentification of residues at these positions that absorb detrimentaleffects to CDR structure from residues located elsewhere in theframework region.

[0040] The frequency of an amino acid residue at a particular frameworkposition is another criteria which can be used for selecting relevantframework amino acid positions to change. For example, comparison of theselected framework with other framework sequences within its subfamilycan reveal residues that occur at minor frequences at a particularposition or positions. Such positions harboring less abundant residuesare similarly applicable for selection as a position to alter in theacceptor variable region framework.

[0041] The relevant amino acid positions to change also can be selected,for example, based on proximity to a CDR. In certain contexts, suchresidues can participate in CDR conformation or antigen binding.Moreover, this criteria can similarly be used to prioritize relevantpositions selected by other criteria described herein. Therefore,differentiating between residues proximal and distal to one or more CDRsis an efficient way to reduce the number of relevant positions to changeusing the methods of the invention.

[0042] Other criteria for selecting relevant amino acid frameworkpositions to alter include, for example, residues that are known orpredicted to reside in three-dimensional space near the antigen-CDRinterface or predicted to modulate CDR activity. Similarly, frameworkresidues that are known or predicted to contact opposite domain of theheavy (V_(H)) and light (V_(L)) chain variable region interface. Suchframework positions can effect the conformation or affinity of a CDR bymodulating the CDR binding pocket, antigen interaction or the V_(H) andV_(L) interaction. Therefore, selection of these amino acid positions asrelevant for construction of the diverse population to screen canbeneficially identify framework changes which replace residues havingdetrimental effects on CDR conformation or absorb detrimental effects ofresidues occurring elsewhere in the framework.

[0043] Finally, other framework residues that can be selected foralteration include amino acid positions that are inaccessible tosolvent. Such residues are generally buried in the variable region andtherefore capable of influencing the conformation of the CDR or V_(H)and V_(L) interactions. Solvent accessibility can be predicted, forexample, from the relative hydrophobicity of the environment created bythe amino acid side chains of the polypeptide or by knownthree-dimensional structural data.

[0044] In addition to selecting the relevant framework positions, themethod of conferring donor CDR binding affinity onto an antibodyacceptor variable region framework also incorporates changes in thedonor CDR amino acid positions. As with selecting the relevant frameworkpositions to change, there is similarly a range of possible changes thatcan be made in the donor CDR positions. Some or all of the possiblechanges that can be selected for change can be introduced into thepopulation of grafted donor CDRs to practice the methods of theinvention.

[0045] One approach is to change all amino acid positions along a CDR byreplacement at each position with, for example, all twenty naturallyoccurring amino acids. The replacement of each position can occur in thecontext of other donor CDR amino acid positions so that a significantportion of the CDR maintains the authentic donor CDR sequence, andtherefore, the binding affinity of the donor CDR. For example, anacceptor variable region framework targeted for relevant amino acidpositions changes as described previously, can be targeted for graftingwith a population of CDRs containing single position replacements ateach position within the CDRS. Similarly, an acceptor variable regionframework can be targeted for grafting with a population of CDRscontaining more than one position changed to incorporate all twentyamino acid residues, or a fractional subset, at each set of positionswithin the CDRs. For example, all possible sets of changes correspondingto two, three or four or more amino acid positions within a CDR can betargeted for introduction into a population of CDRs for grafting into anacceptor variable region framework.

[0046] Single amino acid position changes are generated at each positionwithout altering the remain amino acid positions within the CDR. Apopulation of single position changes will contain at each position thevaried amino acid residues, incorporated either randomly or with abiased frequency, while leaving the remaining positions as donor CDRresidues. For the specific example of a ten residue CDR, the populationwill contain species having the first, second and third, continuedthrough the tenth CDR residue, individually randomized or represented bya biased frequency of incorporated amino acid residues while theremaining non-varied positions represent the donor CDR amino acidresidues. For the specific example described above, these non-variedpositions would correspond to positions 2-10; 1,3-10; 1,2,4-10,continued through positions 1-9, respectively. Therefore, the resultantpopulation will contain species that represent all single positionchanges.

[0047] Similarly, double, triple and quadruple amino acid positionchanges can be generated for each set of positions without altering theremain amino acid positions within the CDR. For example, a population ofdouble position changes will contain at each set of two positions thevaried amino acid residues while leaving the remaining positions asdonor CDR residues. The sets will correspond to, for example, positions1 and 2, 1 and 3, 1 and 4, through the set corresponding to the firstand last position of the CDR. The population will also contain setscorresponding to positions 2 and 3, 2 and 4, 2 and 5, through the setcorresponding to the second an last position of the CDR. Likewise, thepopulation will contain sets of double position changes corresponding toall pairs of position changes beginning with position three of the CDR.Similar pairs of position changes are made with the remaining sets CDRamino acid positions. Therefore, the population will contain speciesthat represent all pairwise combinations of amino acid position changes.In a simialar fashion, populations corresponding to sets of changesrepresenting all triple and quadruplet changes along a CDR can similarlybe targeted for grafting into the variable region frameworks using themethods of the invention.

[0048] The above populations of CDR variant species can be targeted forany or all of the CDRs which constitute the binding pocket of a variableregion. Therefore, an acceptor variable region framework targeted forrelevant amino acid positions changes as described previously, can betargeted for the simultaneous incorporation of donor CDR variantpopulations at one, two or all three recipient CDR locations. The choiceof which CDR or the number of CDRs to target with amino acid positionchanges will depend on, for example, if a full CDR grafting into anacceptor is desired or whether the method is being performed foroptimization of binding affinity. Many grafting procedures willgenerally employ the grafting of all three CDRS, where at least one ofthe CDRs will contain amino acid positions changes. Generally however,all of the donor CDRs will be populations containing amino acid positionchanges. Converesly, and as described further below, optimizationprocedures can employ CDR variant populations corresponding to any orall of the CDRs within a variable region.

[0049] Another approach for selecting donor CDR amino acids to changefor conferring donor CDR binding affinity onto an antibody acceptorvariable region framework is to select known or readily identifiable CDRpositions that are highly variable. For example, the variable region CDR3 is generally highly variable due to genetic recombination. This regiontherefore can be selectively targeted for amino acid position changesduring grafting procedures to ensure binding affinity reacquisition oraugmentation when made together with relevant acceptor variableframework changes as described previously.

[0050] In contrast, CDR residues that appear conserved or have beenempirically determined to be non-mutable by functional criteria willgenerally be avoided when selecting residues in the CDR to target forchange. It should be noted however, that apparent non-mutable residuescan nevertheless be successfully changed using the methods of theinvention because the populations of altered variable regions containfrom a few to many amino acid position changes in both the frameworkregions and in the CDR regions. As such, the CDR grafted variableregions identified by binding affinity are a result of the all thechanges and therefore, all the interactions of residues introduced intoa particular species. Therefore, suboptimal residues incorporated at,for example, an apparent non-mutable position can be counteracted andeven augmented by amino acid substitutions elsewhere in the frameworkregions or in other CDRs.

[0051] Similarly, because the methods of the invention for CDR grafting,affinity reacquisition and affinity optimization employ the productionand screening of diverse populations of variable region speciesgenerated from an acceptor framework and donor CDR variants, there arenumerous effects on binding affinity that will occur due to the combinedinteractions of two or more amino acid changes within a single variableregion species. For example, the affect of amino acid changes in eithera framework region or CDR that are inherently beneficial can be maskedor neutralized due to surrounding authentic parent residues or due totheir context in a heterologous region of a grafted antibody. However,second site changes in the surrounding residues or the heterologousregions can unveil the beneficial characteristics of the latent residueor residues. Such second site changes can occur, for example, in bothproximal and distal heterologous or homologous region sequences.

[0052] For example, if the beneficial residue is in a grafted CDRregion, the proximal heterologous sequences would be the adjacentframework regions whereas distal heterologous regions would be frameworkregions separated by an adjacent CDR. In this specific example, aproximal homologous region would be the surrounding residues within thegrafted CDR harboring the beneficial change whereas the remaining CDRsare examples of distal homologous regions. By analogy, the oppositewould be true for a inherently beneficial residue in a framework region.Specifically, proximal homologous region sequences would be located inthe same framework region and distal homologous sequences would be inany of the other framework regions. Proximal heterologous regionsequences would be in the adjacent CDR or CDRs whereas nonadjacent CDRsconstitute distal heterologous region sequences. Such second siteeffects can occur, for example, through the translation ofconformational changes to the CDR binding pocket or to the frameworkregions.

[0053] Other effects on binding affinity that will occur due to thecombined interactions of two or more amino acid changes within a singlevariable region species include, for example, the neutralization oraugmentation of inherently detrimental changes and the augmentation ofbeneficial amino acid changes or the augmentation of parent residues. Aswith the unveiling of beneficial changes and the ability to counteractchanges in apparently non-mutable residues, the neutralization andaugmentation of amino acid changes within the grafted CDRs or frameworkregion by second site changes can occur, for example, by imparting ortranslating conformational changes from the second site changes to theCDR binding pocket or to the framework regions. The second site changescan occur in any of the framework regions, including for example,framework regions 1 through 4 as well as in any of the three CDRregions. An advantage of the methods of the invention is that no priorinformation is required to assess which amino acid positions or changescan be inherently beneficial or detrimental, or which positions orchanges can be further augmented by second site changes. Instead, byselecting relevant amino acid positions or subsets thereof in theacceptor variable region framework and CDRs, and generating a diversepopulation containing amino acid variants at these positions,combinations of beneficial changes occurring at the selected positionswill be identified by screening for increased or optimized bindingaffinity of the CDR graft variable region. Such beneficial combinationswill include the unveiling of inherently beneficial residues,neutralization of inherently detrimental residues and the augmentationof parent residues or functionally neutral changes.

[0054] Following selection of relevant amino acid positions in theframework regions and in the donor CDRs as described previously, aminoacid changes at some or all of the selected positions are incorporatedinto encoding nucleic acids for the acceptor variable region frameworkand donor CDRs, respectively. Simultaneous with incorporating theencoding amino acid changes at the selected positions, the encodingnucleic acids sequences for each of the donor CDRs, including selectedchanges, are also incorporated into the acceptor variable regionframework encoding nucleic acid to generate a population of alteredvariable region encoding nucleic acids.

[0055] An altered variable region of the invention will contain at leastone framework position which variably incorporates different amino acidresidues and at least one CDR position which variably incorporatesdifferent amino acid residues as described previously. The variabilityat any or all of the altered positions can range from a few to aplurality of different amino acid residues, including all twentynaturally occurring amino acids or functional equivalents and analoguesthereof. The different species of the altered variable region containingthe variable amino acid residues at one or more positions within theframework and CDR regions will make up the population for which toscreen for an altered variable region having binding affinitysubstantially the same or greater than the donor CDR variable region.

[0056] Selection of the number and location of the amino acid positionsto vary is flexible and can depend on the intended use and desiredefficiency for identification of the altered variable region havingsubstantially the same or greater binding affinity compared to the donorvariable region. In this regard, the greater the number of changes thatare incorporated into a altered variable region population, the moreefficient it is to identify at least one species that exhibitssubstantially the same or greater binding affinity as the donor.Alternatively, where the user has empirical or actual data to the affectthat certain amino acid residues or positions contributedisproportionally to binding affinity, then it can be desirable toproduce a limited population of altered variable regions which focuseson changes within or around those identified residues or positions.

[0057] For example, if CDR grafted variable regions are desired, alarge, diverse population of altered variable regions can include allthe non-identical framework region positions between the donor andacceptor framework and all single CDR amino acid position changes.Alternatively, a population of intermediate diversity can includesubsets, for example, of only the proximal non-identical frameworkpositions to be incorporated together with all single CDR amino acidposition changes. The diversity of the above populations can be furtherincreased by, for example, additionally including all pairwise CDR aminoacid position changes. In contrast, populations focusing onpredetermined residues or positions which incorporate variant residuesat as few as one framework and one CDR amino acid position can similarlybe constructed for screening and identification of an altered antibodyvariable region of the invention. As with the above populations, thediversity of such focused populations can be further increased byadditionally expanding on the positions selected for change to includeother relevant positions in either or both of the framework and CDRregions. There are numerous other combinations ranging from few changesto many changes in either or both of the framework regions and CDRs thatcan additionally be employed, all of which will result in a populationof altered variable regions that can be screened for the identificationof at least one CDR grafted altered variable region of the invention.Those skilled in the art will know, or can determine, which selectedresidue positions in the framework or donor CDRs, or subsets thereof,can be varied to produce a population for screening and identificationof a altered antibody of the invention given the teachings and guidanceprovided herein.

[0058] Simultaneous incorporation of all of the CDR encoding nucleicacids and all of the selected amino acid position changes can beaccomplished by a variety of methods known to those skilled in the art,including for example, recombinant and chemical synthesis. For example,simultaneous incorporation can be accomplished by, for example,chemically synthesizing the nucleotide sequence for the acceptorvariable region, fused together with the donor CDR encoding nucleicacids, and incorporating at the positions selected for harboringvariable amino acid residues a plurality of corresponding amino acidcodons.

[0059] One such method well known in the art for rapidly and efficientlyproducing a large number of alterations in a known amino acid sequenceor for generating a diverse population of variable or random sequencesis known as codon-based synthesis or mutagenesis. This method is thesubject matter of U.S. Pat. Nos. 5,264,563 and 5,523,388 and is alsodescribed in Glaser et al. J. Immunology 149:3903 (1992). Briefly,coupling reactions for the randomization of, for example, all twentycodons which specify the amino acids of the genetic code are performedin separate reaction vessels and randomization for a particular codonposition occurs by mixing the products of each of the reaction vessels.Following mixing, the randomized reaction products corresponding tocodons encoding an equal mixture of all twenty amino acids are thendivided into separate reaction vessels for the synthesis of eachrandomized codon at the next position. For the synthesis of equalfrequencies of all twenty amino acids, up to two codons can besynthesized in each reaction vessel.

[0060] Variations to these synthesis methods also exist and include forexample, the synthesis of predetermined codons at desired positions andthe biased synthesis of a predetermined sequence at one or more codonpositions. Biased synthesis involves the use of two reaction vesselswhere the predetermined or parent codon is synthesized in one vessel andthe random codon sequence is synthesized in the second vessel. Thesecond vessel can be divided into multiple reaction vessels such as thatdescribed above for the synthesis of codons specifying totally randomamino acids at a particular position. Alternatively, a population ofdegenerate codons can be synthesized in the second reaction vessel suchas through the coupling of NNG/T nucleotides where N is a mixture of allfour nucleotides. Following synthesis of the predetermined and randomcodons, the reaction products in each of the two reaction vessels aremixed and then redivided into an additional two vessels for synthesis atthe next codon position.

[0061] A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the variant populations described herein.This modification is based on the two vessel method described abovewhich biases synthesis toward the parent sequence and allows the user toseparate the variants into populations containing a specified number ofcodon positions that have random codon changes.

[0062] Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

[0063] Following the above modification of codon-based synthesis,populations containing random codon changes at one, two, three and fourpositions as well as others can be conveniently separated out and usedbased on the need of the individual. Moreover, this synthesis schemealso allows enrichment of the populations for the randomized sequencesover the parent sequence since the vessel containing only the parentsequence synthesis is similarly separated out from the random codonsynthesis.

[0064] Other methods well known in the art for producing a large numberof alterations in a known amino acid sequence or for generating adiverse population of variable or random sequences include, for example,degenerate or partially degenerate oligonucleotide synthesis. Codonsspecifying equal mixtures of all four nucleotide monomers, representedas NNN, results in degenerate synthesis. Whereas partially degeneratesynthesis can be accomplished using, for example, the NNG/T codondescribed previously. Other method well know in the art canalternatively be used such as the use of statistically predetermined, orvarigated, codon synthesis which is the subject matter of U.S. Pat. Nos.5,223,409 and 5,403,484.

[0065] Once the populations of altered variable region encoding nucleicacids have been constructed as described above, they can be expressed togenerate a population of altered variable region polypeptides that canbe screened for binding affinity. For example, the altered variableregion encoding nucleic acids can be cloned into an appropriate vectorfor propagation, manipulation and expression. Such vectors are known orcan be constructed by those skilled in the art and should contain allexpression elements sufficient for the transcription, translation,regulation, and if desired, sorting and secretion of the alteredvariable region polypeptides. The vectors also can be for use in eitherprocaryotic or eukaryotic host systems so long as the expression andregulatory elements are of compatible origin. The expression vectors canadditionally included regulatory elements for inducible or celltype-specific expression. One skilled in the art will know which hostsystems are compatible with a particular vector and which regulatory orfunctional elements are sufficient to achieve expression of thepolypeptides in soluble, secreted or cell surface forms.

[0066] Appropriate host cells, include for example, bacteria andcorresponding bacteriophage expression systems, yeast, avian, insect andmammalian cells. Methods for recombinant expression, screening andpurification of populations of altered variable regions or alteredvariable region polypeptides within such populations in various hostsystems are well known in the art and are described, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1992) and in Ansubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1998). The choice of a particular vector and host system for expressionand screening of altered variable regions will be known by those skilledin the art and will depend on the preference of the user. A specificexample of the expression of recombinant altered variable regionpolypeptides is additionally described below in the Examples. Moreover,expression of diverse populations of hetereomeric receptors in eithersoluble or cell surface form using filamentous bacteriophage vector/hostsystems is well known in the art and is the subject matter of U.S. Pat.No. 5,871,974.

[0067] The expressed population of altered variable region polypeptidescan be screened for the identification of one or more altered variableregion species exhibiting binding affinity substantially the same orgreater than the donor CDR variable region. Screening can beaccomplished using various methods well known in the art for determiningthe binding affinity of a polypeptide or compound. Additionaly, methodsbased on determining the relative affinity of binding molecules to theirpartner by comparing the amount of binding between the altered variableregion polypeptides and the donor CDR variable region can similarly beused for the identification of species exhibiting binding affinitysubstantially the same or greater than the donor CDR variable region.All of such methods can be performed, for example, in solution or insolid phase. Moreover, various formats of binding assays are well knownin the art and include, for example, immobilization to filters such asnylon or nitrocellulose; two-dimensional arrays, enzyme linkedimmunosorbant assay (ELISA), radioimmune assay (RIA), panning andplasmon resonance. Such methods can be found described in, for example,Sambrook et al., supra, and Ansubel et al.

[0068] For the screening of populations of polypeptides such as thealtered variable region populations produced by the methods of theinvention, immobilization of the populations of altered variable regionsto filters or other solid substrate is particularly advantageous becauselarge numbers of different species can be efficiently screened forantigen binding. Such filter lifts will allow for the identification ofaltered variable regions that exhibit substantially the same or greaterbinding affinity compared to the donor CDR variable region.Alternatively, if the populations of altered variable regions areexpressed on the surface of a cell or bacteriophage, for example,panning on immobilized antigen can be used to efficiently screen for therelative binding affinity of species within the population and for thosewhich exhibit substantially the same or greater binding affinity thanthe donor CDR variable region.

[0069] Another affinity method for screening populations of alteredvariable regions polypeptides is a capture lift assay that is useful foridentifying a binding molecule having selective affinity for a ligand(Watkins et. al., (1997)). This method employs the selectiveimmobilization of altered variable regions to a solid support and thenscreening of the selectively immobilized altered variable regions forselective binding interactions against the cognate antigen or bindingpartner. Selective immobilization functions to increase the sensitivityof the binding interaction being measured since initial immobilizationof a population of altered variable regions onto a solid support reducesnon-specific binding interactions with irrelevant molecules orcontaminants which can be present in the reaction.

[0070] Another method for screening populations or for measuring theaffinity of individual altered variable region polypeptides is throughsurface plasmon resonance (SPR). This method is based on the phenomenonwhich occurs when surface plasmon waves are excited at a metal/liquidinterface. Light is directed at, and reflected from, the side of thesurface not in contact with sample, and SPR causes a reduction in thereflected light intensity at a specific combination of angle andwavelength. Biomolecular binding events cause changes in the refractiveindex at the surface layer, which are detected as changes in the SPRsignal. The binding event can be either binding association ordisassociation between a receptor-ligand pair. The changes in refractiveindex can be measured essentially instantaneously and therefore allowsfor determination of the individual components of an affinity constant.More specifically, the method enables accurate measurements ofassociation rates (k_(on)) and disassociation rates (k_(off)).

[0071] Measurements of k_(on) and k_(off) values can be advantageousbecause they can identify altered variable regions or optimized variableregions that are therapeutically more efficacious. For example, analtered variable region, or heteromeric binding fragment thereof, can bemore efficacious because it has, for example, a higher k_(on) valuedcompared to variable regions and heteromeric binding fragments thatexhibit similar binding affinity. Increased efficacy is conferredbecause molecules with higher k_(on) values can specifically bind andinhibit their target at a faster rate. Similarly, a molecule of theinvention can be more efficacious because it exhibits a lower k_(off)value compared to molecules having similar binding affinity. Increasedefficacy observed with molecules having lower k_(off) rates can beobserved because, once bound, the molecules are slower to dissociatefrom their target. Although described with reference to the alteredvariable regions and optimized variable regions of the inventionincluding, heteromeric variable region binding fragments thereof, themethods described above for measuring associating and disassociationrates are applicable to essentially any antibody or fragment thereof foridentifying more effective binders for therapeutic or diagnosticpurposes.

[0072] Methods for measuring the affinity, including association anddisassociation rates using surface plasmon resonance are well known inthe arts and can be found described in, for example, Jonsson andMalmquist, Advances in Biosnsors, 2:291-336 (1992) and Wu et al. Proc.Natl. Acad. Sci. USA, 95:6037-6042 (1998). Moreover, one apparatus wellknown in the art for measuring binding interactions is a BIAcore 2000instrument which is commercially available through Pharmacia Biosensor,(Uppsala, Sweden).

[0073] Using any of the above described screening methods, as well asothers well known in the art, an altered variable region having bindingaffinity substantially the same or greater than the donor CDR variableregion is identified by detecting the binding of at least one alteredvariable region within the population to its antigen or cognate ligand.Additionally, the above methods can alternatively be modified by, forexample, the addition of substrate and reactants, to identify using themethods of the invention, altered variable regions having catalyticactivity substantially the same or greater that the donor CDR variableregion within the populations. Comparision, either independently orsimultaneously in the same screen, with the donor variable region willidentify those binders that have substantially the same or greaterbinding affinity as the donor. Those skilled in the art will know, orcan determine using the donor variable region, binding conditions whichare sufficient to identify selective interactions over non-specificbinding.

[0074] Detection methods for identification of binding species withinthe population of altered variable regions can be direct or indirect andcan include, for example, the measurement of light emission,radioisotopes, calorimetric dyes and fluorochromes. Direct detectionincludes methods that operate without intermediates or secondarymeasuring procedures to assess the amount of bound antigen or ligand.Such methods generally employ ligands that are themselves labeled by,for example, radioactive, light emitting or fluorescent moieties. Incontrast, indirect detection includes methods that operate through anintermediate or secondary measuring procedure. These methods generallyemploy molecules that specifically react with the antigen or ligand andcan themselves be directly labeled or detected by a secondary reagent.For example, a antibody specific for a ligand can be detected using asecondary antibody capable of interacting with the first antibodyspecific for the ligand, again using the detection methods describedabove for direct detection. Indirect methods can additionally employdetection by enzymatic labels. Moreover, for the specific example ofscreening for catalytic antibodies, the disappearance of a substrate orthe appearance of a product can be used as an indirect measure ofbinding affinity or catalytic activity.

[0075] Isolated variable regions exhibit binding affinity as singlechains, in the absence of assembly into a heteromeric structure withtheir respective V_(H) or V_(L) subunits. As such, populations of V_(H)and V_(L) altered variable regions polypeptides can be expressed aloneand screened for binding affinity having substantially the same orgreater binding affinity compared to the CDR donor V_(H) or V_(L)variable region. Alternatively, populations of V_(H) and V_(L) alteredvariable regions polypeptides can be coexpressed so that theyself-assemble into heteromeric altered variable region bindingfragments. The heteromeric binding fragment population can then bescreened for species exhibiting binding affinity substantially the sameor greater than the CDR donor variable region binding fragment. Aspecific example of the coexpression and self-assembly of populationsV_(H) and V_(L) altered variable regions into hetermeric populations isdescribed further below in the Examples.

[0076] Therefore, the invention provides a method of simultaneouslygrafting and optimizing the binding affinity of a variable regionbinding fragment. The method consists of: (a) constructing a populationof altered heavy chain variable region encoding nucleic acids consistingof an acceptor variable region framework, containing donor CDRs and aplurality of different amino acids at one or more framework region andCDR amino acid positions; (b) coexpressing the populations of heavy andlight chain variable region encoding nucleic acids to produce diversecombinations of heteromeric variable region binding fragments, and (c)identifying one or more heteromeric variable region binding fragmentshaving affinity substantially the same or greater than the donor CDRheteromeric variable region binding fragment.

[0077] The invention additionally provides a method of optimizing thebinding affinity of an antibody variable region. The consists of: (a)constructing a population of antibody variable region encoding nucleicacids, said population comprising two or more CDRs containing aplurality of different amino acids at one or more CDR amino acidpositions; (b) expressing said population of variable region encodingnucleic acids, and (c) identifying one or more variable regions havingbinding affinity substantially the same or greater than the donor CDRvariable region.

[0078] The methods described above, for conferring donor CDR bindingaffinity onto an antibody acceptor variable region framework and forsimultaneously grafting and optimizing the binding affinity of aheteromeric variable region binding fragment, can additionally beemployed to modify or optimize the binding affinity of a variable regionor a heteromeric variable region binding fragment. Similar to thepreviously described methods, the method for modifying or optimizingbinding affinity involves the selection of relevant amino acid positionsand the construction, expression and screening of variable regionpopulations containing variable amino acid residues at all or a fractionof the selected positions. However, for optimization of binding affinityit is not necessary to vary amino acid positions in the frameworkregions. Instead, all that is required is to alter one or more aminoacid positions in two or more CDR regions. Changing the CDR amino acidresidues directly effects the binding affinity. Once a populationcontaining variable amino acid residues incorporated in two or more CDRsis produced, all that is necessary is to screen the population forspecies that contain the desired binding affinity modification. All ofthe criteria for selecting relevant amino acid positions describedpreviously are applicable for use in this mode of the method. Therefore,the methods for modifying or optimizing the binding affinity of avariable region or a heteromeric variable region binding fragment byaltering one or more amino acid positions in two or more CDR regions areapplicable to essentially any variable region, grafted variable regionas well as applicable to the altered and optimized variable regions ofthe invention.

[0079] Moreover, by incorporating variable amino acid residues in two ormore CDRs when employing the methods conferring donor CDR bindingaffinity onto an acceptor framework, this method of modifying bindingaffinity is therefore useful for simultaneously optimizing the bindingaffinity of a grafted antibody. Employing the methods for simultaneouslygrafting and optimizing, or for optimizing, it is possible to generateheteromeric variable region binding fragments having increases inaffinities of greater than 5-, 8- and 10-fold. In particular,heteromeric variable region binding fragments can be generated havingincreases in affinities of greater than 12-, 15-20- and 25-fold as wellas affinities greater than 50-, 100- and 1000-fold compared to the donoror parent molecule.

[0080] Additionally, the methods described herein for optimizing arealso are applicable for producing catalytic heteromeric variable regionfragments or for optimizing their catalytic activity. Catalytic activitycan be optimized by changing, for example, the on or off rate, thesubstrate binding affinity, the transition state binding affinity, theturnover rate (kcat) or the Km. Methods for measuring thesecharacteristics are well known in the art. Such methods can be employedin the screening steps of the methods described above when used foroptimizing the catalytic activity of a heteromeric variable regionbinding fragment.

[0081] The methods for conferring donor CDR binding affinity onto anantibody acceptor variable region framework described previously areapplicable for use with essentially any distinguishable donor andacceptor pair. Many applications of the methods will be for theproduction and optimization of variable region binding fragments havinghuman acceptor frameworks due to the therapeutic importance of suchmolecules in the treatment of human diseases. However, the method areapplicable for conferring donor CDR binding affinity onto an acceptororiginating from the same or a divergent species as the CDR donorvariable region so long as the framework regions between the donor andacceptor variable regions are distinct. Therefore, the inventionincluded altered variable regions having acceptor frameworks derivedfrom human, mouse, rat, rabbit, goat and chicken, for example.

[0082] Additionally, the methods for conferring donor CDR bindingaffinity onto an antibody acceptor variable region framework areapplicable for grafting CDRs as described by Kabat et al., supra,Chothia et al., supra or MacCallum et al., supra. The methods similarlycan be used for grafting into an acceptor framework overlapping regionsor combinations of CDR as described by these authors. Generally, themethods will graft variable region CDRs by identifying the boundriesdescribed by one of the CDR definitions known in the art and set forthherein. However, because the methods are directed to constructing andscreening populations of CDR grafted altered variable regions whichincorporate relevant amino acid position changes in both the frameworkand CDR regions, and such variations can, for example, compensate oraugment amino acid changes elsewhere in the variable region, the exactboundry of a particular CDR or set of variable region CDRs can bevaried. Therefore, the exact CDR region to graft, whether it is theregion described by Kabat et al., Chothia et al. or MacCallum et al., orany combination thereof, will essentially depend on the preference ofthe user.

[0083] Similarly, the methods described previously for optimizing thebinding affinity of an antibody also are applicable for use withessentially any variable region for which an encoding nucleic acid is,or can be made available. As with the methods for conferring donor CDRbinding affinity, many applications of the methods for optimizingbinding affinity will be for modifying the binding affinity of CDRgrafted variable regions having human frameworks. Again, such moleculesare significantly less antigenic in human patients and thereforetherapeutically valuable in the treatment of human diseases. However,the methods of the invention for optimizing the binding affinity of avariable region are applicable to all species of variable regions.Therefore, the invention includes binding affiity optimization ofvariable regions derived from human, mouse, rat, rabbit, goat andchicken, for example.

[0084] The methods of the invention have been described with referenceto variable regions and heteromic variable region binding fragments.Given these descriptions and teaching herein, those skilled in the artwill understand that all of such methods are applicable to wholeantibodies and functional fragments thereof as well as to regions andfunctional domains other than the antigen binding variable region ofantibodies. Moreover, the methods described herein are furtherapplicable to molecules other than antibodies, variable regions andother antibody functional domains. Given the teachings of the invention,those skilled in the art will know how to apply the methods ofsimultaneously constructing hybrid molecules and maintaining oroptimizing the binding affinity or catalytic activity of a targetmolecule, as well as how to apply the methods of optimizing the bindingaffinity or catalytic activity to a variety of different types andclasses of polypeptides and proteins.

[0085] It is understood that modifications which do not substantiallyaffect the activity of the various embodiments of this invention arealso included within the definition of the invention provided herein.Accordingly, the following examples are intended to illustrate but notlimit the present invention.

EXAMPLE I Simultaneous Humanization and Affinity Maturation of anAnti-CD40 Antibody

[0086] This example shows the simultaneous humanization and affinitymaturation of the murine mAb 40.2.220, directed against the CD40receptor.

[0087] The CD40 receptor is a potential therapeutic target for severaldiseases. For example, the interaction of the CD40 receptor and itsligand, gp39, serves a critical role in both humoral and cell-mediatedimmune responses (Foy et. al., (1996)). Immunological rejection oforgans from genetically non-identical individuals, termedgraft-versus-host-disease (GVHD), is mediated through T cell-dependentmechanisms. In vivo administration of an anti-gp39 mAb blocks GVHD inmice and inhibits many of the GVHD-associated phenomena (Durie et. al.,1994)), providing evidence that the CD40/gp39 interaction plays acritical role in the development of GVHD. More recently, inhibition ofthe CD40/gp39 interaction in vivo in hyperlipidemic mice fed a highcholesterol diet limited atherosclerosis, suggesting that CD40signalling may also play a role in atherogenesis (Mach et. al., (1998)).In addition, the CD40 receptor is overexpressed on hematologicmalignancies (Uckun et. al., 1990)) and certain carcinomas (Stamenkovicet. al., (1989)) and thus, may serve as a target for cytotoxic agents.An anti-CD40 single chain antibody-toxin fusion was cytotoxic againstCD40-expressing malignant cells in vitro (Francisco et. al., (1995)) andwas efficacious in treating human non-Hodgkin's lymphoma xenograftedSCID mice (Francisco et. al., (1997)).

[0088] Codon-based mutagenesis (Glaser et. al., (1992)) was used tocreate libraries of LCDR3, HCDR3 and framework region variants of mAb40.2.220 sequences. Libraries composed of framework region variantsalone and in combination with HCDR3 variants and with HCDR3 and LCDR3variants together were screened for high affinity variants. It wasdemonstrated that in combination higher affinity variants were obtainedthan those obtained when codon-based mutagenesis was appliedindependently thus showing (1) higher affinity variants that could onlybe obtained by the use of codon-based mutagenesis simultaneously ondisparate regions of the mAb and (2) the use of codon-based mutagenesisto uncover potential direct interactions between disparate regions of amAb.

[0089] A vector for the production of a chimeric anti-CD40 murine mAb40.2.220 was constructed. Based on the sequence of anti-CD40 murine mAb40.2.220 (provided by Dr. D. Hollenbaugh, Bristol-Myers Squibb,Princeton, N.J.) overlapping oligonucleotides encoding V_(H) and V_(L)(69-75 bases in length) were synthesized and purified. The variable Hand L domains were synthesized separately by combining 25 pmol of eachof the overlapping oligonucleotides with Pfu DNA polymerase (Stratagene)in a 50 μl PCR reaction consisting of 5 cycles of: denaturing at 94° C.for 20 sec, annealing at 50° C. for 30 sec, ramping to 72° C. over 1min, and maintaining at 72° C. for 30 sec. Subsequently, the annealingtemperature was increased to 55° C. for 25 cycles. A reverse primer anda biotinylated forward primer were used to further amplify 1 μl of thefusion product in a 100 μl PCR reaction using the same program. Theproducts were purified by agarose gel electrophoresis, electroeluted,and phosphorylated by T4 polynucleotide kinase (Boehringer Mannheim) andwere then incubated with streptavidin magnetic beads (BoehringerMannheim) in 5 mM Tris-Cl, pH 7.5, 0.5 mM EDTA, 1 M NaCl, and 0.05%Tween 20 for 15 min at 25° C. The beads were washed and thenon-biotinylated, minus strand DNA was eluted by incubating with 0.15 MNaOH at 25° C. for 10 min. Chimeric anti-CD40 Fab was synthesized in amodified M131X104 phage vector (Kristensson et. al., 1995)), termedM131X104CS, by hybridization mutagenesis (Rosok et. al., (1996); Kunkel,(1985)) using the V_(H) and V_(L) oligonucleotides in 3-fold molarexcess of the uridinylated vector template. The M131X104 vector wasmodified by replacing cysteine residues at the end of the kappa and ylconstant regions with serine. The reaction was electroporated into DH10Bcells and titered onto a lawn of XL-1 Blue.

[0090] The murine anti-CD40 mAb variable region framework sequences wereused to identify the most homologous human germline sequences. The Hchain framework residues were 74% identical to human germline VH7(7-4.1) and JH4 sequences while the L chain was 75% identical to thecorresponding human germline VKIII (L6) and JK4 sequences. Alignment ofthe H and L chain variable sequences is shown in FIG. 1. CDR residues,as defined by Kabat et. al. (1977, 1991), are underlined and wereexcluded from the homology analysis. Differences in sequence areindicated by vertical lines and framework positions characterized in thecombinatorial expression library are marked with an asterisk. Frameworkresidues that differed between the murine mAb and the human templateswere assessed individually.

[0091] Based on structural and sequence analysis, antibody CDRs with theexception of HCDR3 display a limited number of main chain conformationstermed canonical structures (Chothia & Lesk, (1987); Chothia et. al.,(1989)). Moreover, certain residues critical for determining the mainchain conformation of the CDR loops have been identified (Chothia &Lesk, (1987); Chothia et. al., (1989)). Canonical framework residues ofmurine anti-CD40 were identified therefore, and it was determined thatamino acids at all critical canonical positions within the H and L chainframeworks of the human templates were identical to the correspondingmurine residues.

[0092] Surface-exposed murine amino acids not normally found in humanantibodies are likely to contribute to the immunogenicity of thehumanized mAb (Padlan, (1991)). Therefore, framework residues differingbetween murine anti-CD40 and the human templates were analyzed and basedon solvent exposure were predicted to be buried or located on thesurface of the antibody (Padlan, (1991)). Solvent-exposed frameworkresidues distal to the CDRs were not expected to contribute to antigenbinding significantly and thus, with the exception of two H chainresidues all were changed to the corresponding human amino acid todecrease potential immunogenicity. H chain residues 28 and 46 werepredicted to be solvent exposed. However, H28 is located within theHCDR1 region as defined by Chothia & Lesk (1987) and potentiallyinteracts with the antigen. In addition, the lysine at H46 in the murinemAb is somewhat unusual and significantly different from the glutamicacid of the human template. Therefore, the murine and human residues atH28 and H46 were expressed in the combinatorial library (FIG. 1,asterisks).

[0093] The remaining differing framework residues, all predicted to bemostly buried within the antibody, were evaluated for: (1) proximity toCDRs, (2) potential to contact the opposite domain in the V_(K)-V_(H)interface, (3) relatedness of the differing amino acids, and (4)predicted importance in modulating CDR activity as defined by Studnickaet. al., (1994). The majority of L chain framework differences in buriedresidues were related amino acids at positions considered not likely tobe directly involved in the conformation of the CDR. However, L49 islocated adjacent to LCDR2, potentially contacts the V_(H) domain, isunrelated to the human residue, and may be involved in determining theconformation of LCDR2. For these reasons, the murine and human aminoacids at L49 were both expressed in the combinatorial framework library(FIG. 1, asterisk).

[0094] Analysis of the murine H chain sequence and the human templatewas performed. Residue H9 is a proline in the murine mAb while the humantemplate contains an unrelated serine residue. Position H9 can also playa role in modulating the conformation of the CDR and thus, was selectedas a combinatorial library site (FIG. 1, asterisks). The remainingburied framework residues that differed between murine anti-CD40 and theH chain template were at framework positions 38, 39, 48, and 91. Murineanti-CD40 mAb contained glutamine and glutamic acid at H38 and H39,respectively, while the human template contained arginine and glutamine.Residue H38 is in proximity to the HCDR1, the glutamine→arginine changeis non-conserved, and expression of glutamine at this site in murine Absis somewhat unusual. Similarly, glutamic acid→glutamine is anon-conservative difference for buried amino acids, H39 is a potentialV_(K) contact residue, and glutamic acid is somewhat unusual in murinemAbs. Residue H48 is in close proximity to HCDR2 and H91 is predicted tobe a high risk site (Studnicka et. al., (1994); Harris & Bajorath,(1995)) that potentially contacts the V_(K) domain. Thus, both murineand human residues were expressed at H38, 39, 48, and 91 (FIG. 1,asterisks).

[0095] The combinatorial framework library (Hu I) was synthesized by thesame method used to construct the chimeric anti-CD40, withmodifications. Overlapping oligonucleotides encoding the frameworkregions of the H and L variable domains of the human template and themurine anti-CD40 CDRs as defined by Kabat et. al. (1977, 1991) weresynthesized. Among these, degenerate oligonucleotides encoding both themurine and the human amino acids at seven V_(H) and one V_(K) frameworkposition as selected above were synthesized (FIG. 1 residues marked withasterisk). All of these sites were characterized by synthesizing acombinatorial library that expressed all possible combinations of themurine and human amino acids found at these residues. The totaldiversity of this library, termed Hu I, was 2⁸ or 256 variants (TableI).

[0096] The Hu I combinatorial library was first screened by an ELISAthat permits the rapid assessment of the relative affinities of thevariants (Watkins et. al., (1997)). Briefly, microtiter plates werecoated with 5 μg/ml goat anti-human kappa (Southern Biotechnology) andblocked with 3% BSA in PBS. Certain Fabs were cloned into an expressionvector under the control of the arabinose-regulated BAD promoter. Inaddition, a six-histidine tag was fused to the carboxyl-terminus of theH chain to permit purification with nickel-chelating resins. PurifiedFab was quantitated as described (Watkins et. al., 1997). Next, 50 μlFab from the Escherichia coli culture supernatant or from the celllysate, was incubated with the plate 1 h at 25° C., the plate was washedthree times with PBS containing 0.1% Tween 20, and incubated with 0.1μg/ml CD40-Ig in PBS containing 1% BSA for 2 h at 25° C. The plate waswashed three times with PBS containing 0.1% Tween 20 and goat anti-mouseIgG_(2b)-alkaline phosphatase conjugate diluted 3000-fold in PBScontaining 1% BSA was added for 1 h at 25° C. The plate was washed threetimes with PBS containing 0.1% Tween 20 and was developed as described(Watkins et. al., (1997)).

[0097] Although variants that bind the target antigen with affinitiescomparable to, or better than the chimeric Fab were identified, themajority of Hu I clones screened were less active than the chimericanti-CD40 Fab. Approximately 6% of randomly selected Hu I variantsdisplayed binding activities comparable to the chimeric Fab (data notshown). The identification of multiple Hu I variants with activitycomparable to the chimeric CD40 is consistent with the interpretationthat the most critical framework residues were included in thecombinatorial library.

[0098] The kinetic constants for the interaction between CD40 and theanti-CD40 variants were determined by surface plasmon resonance(BIAcore). CD40-Ig fusion protein was immobilized to a(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride) andN-hydroxysuccinimide-activated sensor chip CM5 by injecting 8 μl of 10μg/ml CD40-Ig in 10 mM sodium acetate, pH 4. CD40-Ig was immobilized ata low density (˜150 RU) to prevent rebinding of Fabs during thedissociation phase. To obtain association rate constants (k_(on)), thebinding rate at six different Fab concentrations ranging from 25-600 nMin PBS was determined at a flow rate of 20 μl/min. Dissociation rateconstants (k_(off)) were the average of six measurements obtained byanalyzing the dissociation phase. Sensorgrams were analyzed with theBIAevaluation 3.0 program. K_(d) was calculated fromK_(d)=k_(off)/k_(on), Residual Fab was removed after each measurement byprolonged dissociation.

[0099]FIG. 2A shows bacterially-expressed chimeric anti-CD40 Fab andcertain variants from each of the libraries were titrated on immobilizedantigen. Chimeric (filled circles), Hu I-19C11 (open circles), HuII-CW43 (open squares), Hu III-2B8 (filled triangles), and an irrelevant(filled squares) Fab were released from the periplasmic space of 15 mlbacterial cultures and serial dilutions were incubated with CD40-Igantigen immobilized on microtiter plates. See below for description ofHuII and HuIII libraries. Antibody binding was quantitated as describedabove. These measurements confirm the identification of multiplevariants with enhanced affinity. For example, clone 19C11 binds the CD40receptor with higher affinity than the chimeric Fab, as demonstrated bythe shift in the titration profile (compare open circles with filledcircles). DNA sequencing of 34 of the most active clones led to theidentification of 24 unique framework combinations, each containing 2-6murine framework residues (data not shown).

[0100] LCDR3 and HCDR3 contact antigen directly, interact with the otherCDRs, and often affect the affinity and specificity of antibodiessignificantly (Wilson & Stanfield, (1993); Padlan, (1994)). In addition,the conformation of LCDR3 and HCDR3 are determined in part by certainframework residues. Therefore, to identify the most active antibody itcould be beneficial to construct combinatorial libraries that optimizethe third CDR of the H and L chains in conjunction with selecting themost active framework.

[0101] Codon-based mutagenesis (Glaser et. al., (1992)) was used tosynthesize oligonucleotides that introduce mutations at every positionin HCDR3, one at a time, resulting in the expression of all 20 aminoacids at each CDR residue from Ser⁹⁵-Tyr¹⁰² (FIG. 1, underlined).Briefly, for library construction, the overlapping oligonucleotidesencoding the framework library and non-library murine CDR were combinedwith 25 pmol of the oligonucleotides encoding mutated HCDR3. The pool ofoligonucleotides encoding the HCDR3 library was mixed with theoverlapping oligonucleotides encoding the combinatorial framework andother CDRs to generate a framework/HCDR3 library. The diversity of thislibrary, termed Hu II, was 1.1×10⁵ (Table I).

[0102] The CDR residues selected for mutagenesis of LCDR3 wereGln⁸⁹-Thr⁹⁷ (FIG. 1, underlined). Oligonucleotides encoding LCDR3 weredesigned to mutate a single CDR residue in each clone as described abovefor HCDR3. Oligonucleotides encoding the LCDR3, HCDR3, and thecombinatorial framework were used to create a framework/HCDR3/LCDR3library, termed Hu III. The large number of framework/CDR3 combinationsresulted in a library with a complexity of 3.1×10⁷ (Table I). TABLE ISummary of phage-expressed anti-CD40 antibody libraries. Library LibraryPositions Size* Screened^(†) Hu I framework 256 2.4 × 10³ Hu IIframework, HCDR3 1.1 × 2.0 × 10⁶ 10⁵ Hu III framework, HCDR3, 3.1 × 5.5× 10⁵ LCDR3 10⁷

[0103] An additional library (Hu IV) was synthesized to further optimizethe best variant (clone F4) identified from the Hu III library.Oligonucleotides encoding LCDR3, designed to mutate a single CDR residuein each clone, were synthesized by introducing NN(G/T) at each position(Glaser et. al., (1992)) and were annealed to uridinylated F4 template(Kunkel, (1985)) which already contained a ⁹⁶R→W mutation in LCDR3.

[0104] Combining mutations in LCDR3 and/or HCDR3 with the frameworklibrary increased the potential diversity of humanized anti-CD40variants from 256 to greater than 10⁷. In order to screen these largerlibraries more efficiently a modified plaque lift assay, termed capturelift, was used (Watkins et. al., (1997)). Briefly, nitrocellulosefilters (82-mm) were coated with goat anti-human kappa, blocked with 1%BSA, and were applied to an agar plate containing the phage-infectedbacterial lawn. In the initial screen, phage were plated at 10⁵phage/100-mm plate. After the capture of phage-expressed anti-CD40variant Fabs, the filters were incubated 3 h at 25° C. with 5 ng/mlCD40-Ig in PBS containing 1% BSA. The filters were rinsed four timeswith PBS containing 0.1% Tween 20 and were incubated with goatanti-mouse IgG_(2b)-alkaline phosphatase conjugate (SouthernBiotechnology) diluted 3000-fold in PBS containing 1% BSA for 1 h at 25°C. The filters were washed four times with PBS containing 0.1% Tween 20and were developed as described (Watkins et. al., (1998)). To isolateindividual clones, positive plaques from the initial screen were picked,replated at lower density (<10³ phage/100-mm plate), and were screenedby the same approach. Because the filters were probed with antigen at aconcentration substantially below the Kd of the Fab only variantsdisplaying enhanced affinity were detectable. Multiple clones displayinghigher affinities were identified following the screening of >10⁶variants from Hu II and >10⁵ variants from the Hu III library using82-mm filters containing 10⁵ variants per filter (Table I). Titration ofthe variants on immobilized CD40-Ig verified that multiple clonesdisplayed affinities greater than the chimeric and humanized Fab (FIG.2A, compare open squares, filled triangles with circles).

[0105] The framework/CDR mutations that conferred enhanced affinity wereidentified by DNA sequencing. Single-stranded DNA was isolated and the Hand L chain variable region genes were sequenced by the fluorescentdideoxynucleotide termination method (Perkin-Elmer, Foster City,Calif.). Unique variable region sequences were identified in 10/13 Hu IIvariants and 4/5 Hu III variants. Both the Hu II and Hu III variantscontained 1-5 murine framework residues and 0-2 CDR3 mutations.Representative examples are summarized in Table II. The affinities ofbacterially-expressed chimeric Fab and certain variants from each of thelibraries were characterized more thoroughly using surface plasmonresonance measurements to determine the association and dissociationrates of purified Fab with immobilized CD40-Ig as described above.

[0106] Chimeric anti-CD40 had a dissociation constant K_(d)=48.3 nM and,consistent with the screening results, the variants all displayed higheraffinities with K_(d) ranging from 0.24 nM to 10.5 nM (Table II).Further optimization of LCDR3 of Hu III clone F4 resulted in theidentification of a higher affinity (K_(d)=0.1 nM) clone, L3.17, whichcontained a 94 F→Y mutation. The improved affinities of the anti-CD40variants were predominantly the result of slower dissociation rates.However, the association rates of most variants were also enhanced,increasing by as much as ≈3-fold (1.2 vs. 3.2×10⁶ M⁻¹s⁻¹ for chimericanti-CD40 and clone L3.17, respectively). TABLE II Simultaneousoptimization of framework and CDR residues.

# a single differing L chain (L) framework residue as position 49.

[0107] The variants displaying enhanced affinity were tested for theirability to block the binding of gp39 ligand to the CD40 receptor.Immulon II microtiter plates were coated with 2 μg/ml anti-murine CD8 tocapture sgp39 fusion protein which expresses the CD8 domain. The plateswere rinsed once with PBS containing 0.05% Tween 20, and were blockedwith 3% BSA in PBS. The plate was washed once with PBS containing 0.05%Tween 20 and was incubated with cell culture media containing saturatinglevels of sgp39 for 2 h at 25° C. Unbound sgp39 was aspirated and theplate was washed two times with PBS containing 0.05% Tween 20. Next, 25μl of purified variant Fabs diluted serially 3-fold in PBS was addedfollowed by 25 μl of 4 μg/ml CD40-human Ig in PBS. The plates wereincubated 2 h at 25° C. and were washed three times with PBS containing0.05% Tween 20. Bound CD40-human Ig was detected following a 1 hincubation at 25° C. with goat F(ab′)₂ anti-human IgG Fcγ-specifichorseradish peroxidase conjugate (Jackson) diluted 10,000-fold in PBS.The plate was washed four times with PBS containing 0.05% Tween 20 andbinding was quantitated calorimetrically by incubating with 1 mg/mlo-phenylenediamine dihydrochloride and 0.003% hydrogen peroxide in 50 mMcitric acid, 100 mM Na₂HPO₄, pH 5. The reaction was terminated by theaddition of H₂SO₄ to a final concentration of 0.36 M and the absorbanceat 490 nm was determined. FIG. 2B shows purified variants were testedfor their ability to inhibit sgp39 binding to CD40-Ig. The ligand forthe CD40 receptor, gp39, was captured in a microtiter plate andsubsequently, varying amounts of purified chimeric (filled circles), Hu11-CW43 (open squares), Hu III-2B8 (filled triangles), Hu II/III-2B12(open triangles), and irrelevant (filled squares) Fab were co-incubatedwith 2 μg/ml CD40-human Ig on the microtiter plate. The variants allinhibited the binding of soluble CD40-Ig fusion protein to immobilizedgp39 antigen in a dose-dependent manner that correlated with theaffinity of the Fabs. For example, one of the most potent inhibitors ofligand binding to CD40-Ig fusion protein was variant 2B8, which was alsoone of the variants with the highest affinity for CD40. Variant 2B8displayed ≈17-fold higher affinity for CD40 than did the chimeric Faband inhibited ligand binding ≈7-fold more effectively.

[0108] Screening of the Hu I library identified two variants that weresimilar or identical in framework sequence to the Hu II clone CW43 butdisplayed 5-fold lower affinities (Table II, clones 1H11 and 9A3). Clone9A3 has an identical framework structure while 1H11 contained the murinelysine framework residue at L chain position 49. Sequence comparisonsand site-directed mutagenesis studies (data not shown) suggest that thebeneficial arginine residue at HCDR3 position 101 might interact with Lchain residue tyr⁴⁹. To test this, L chain residue tyr⁴⁹ of clone CW43was mutated to the lysine murine framework residue, resulting in avariant with a framework identical to clone 1H11 that also contained thebeneficial arg¹⁰¹ residue in HCDR3. The resulting mAb, termed Y49K,displayed 5-fold lower affinity than CW43. Thus, expression of tyrosineat L chain framework residue 49 or expression of arginine at HCDR3residue 101 alone had no beneficial effect on the mAb affinity, whilethe concomitant expression of tyrosine and arginine at these sitesimproved the mAb affinity 5-fold. The non-additive, or dependent natureof the mutations demonstrates that L chain residue tyr⁴⁹ and HCDR3residue arg¹⁰¹ interact co-operatively to enhance the affinity of themAb (Schreiber & Fersht, (1995)). In addition, the co-operativeinteraction that was observed between tyr⁴⁹ and arg¹⁰¹ was also observedfor variants that expressed lysine at HCDR3 position 101 (Table II).

[0109] Generally, interacting residues are spatially separated by nomore than 7 A (Schreiber & Fersht, 1995)). FIG. 3 shows molecularmodeling of anti-CD40 variant CW43. A top view of the anti-CD40 variantCW43 variable region structure was created by homology modeling toexamine the spatial relationship of L chain framework residue Y49 and Hchain CDR3 residue R101. The L chain is on the left and the H chainright with the H chain CDR3 loop depicted in red. The L chain frameworkresidue 49 is in close proximity to the H chain CDR3 loop and is 7 Å ofthe predicted interacting H chain CDR3 R101 residue. Although theinteracting amino acids are located on distinct chains of the mAb, theresidues are predicted to be within a range (7 Å) to permit co-operativeinteraction.

[0110] Throughout this application various publications have beenreferenced within parentheses. The disclosures of these publications intheir entireties are hereby incorporated by reference in thisapplication in order to more fully describe the state of the art towhich this invention pertains.

[0111] Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1 4 1 107 PRT Mus musculus 1 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr LeuSer Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala SerGln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His GluSer Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser His Ser Ile Ser Gly IlePro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Ser Asp Phe Thr Leu SerIle Asn Ser Val Glu Pro 65 70 75 80 Glu Asp Val Gly Ile Tyr Tyr Cys GlnHis Gly His Ser Phe Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu GluIle Lys 100 105 2 107 PRT Homo sapiens 2 Glu Ile Val Leu Thr Gln Ser ProAla Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln LysPro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg AlaThr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val TyrTyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly ThrLys Val Glu Ile Lys 100 105 3 122 PRT Mus musculus 3 Gln Ile Gln Leu ValGln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Arg IleSer Cys Lys Ala Ser Gly Tyr Ala Phe Thr Thr Thr 20 25 30 Gly Met Gln TrpVal Gln Glu Met Pro Gly Lys Gly Leu Lys Trp Ile 35 40 45 Gly Trp Ile AsnThr His Ser Gly Val Pro Lys Tyr Val Glu Asp Phe 50 55 60 Lys Gly Arg PheAla Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Leu Gln IleSer Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Val Arg SerGly Asn Gly Asn Tyr Asp Leu Ala Tyr Phe Ala Tyr Trp 100 105 110 Gly GlnGly Thr Leu Val Thr Val Ser Ala 115 120 4 113 PRT Homo sapiens 4 Gln ValGln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 SerVal Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 AlaMet Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 GlyTrp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe 50 55 60 ThrGly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105110 Ser

1. A method of constructing a population of altered heavy chain variableregion encoding nucleic acids, comprising: a) providing a representationof first and second reference amino acid sequences, said first referencesequence comprising the sequence of a donor heavy chain variable region,said donor variable region comprising i) framework regions and ii) threecomplementarity-determining regions as defined by the combineddefinitions of Kabat and Chothia; said second reference sequencecomprising the sequence of an acceptor heavy chain variable regioncomprising framework regions; b) synthesizing a) first oligonucleotidesencoding portions of said framework regions of said acceptor heavy chainvariable region, wherein said portions of said framework regions whencompared to said second reference sequence are unmodified; and b) apopulation of second oligonucleotides, each encoding i) at least aportion of a first complementarity-determining region that has beenmodified, said first complementarity-determining region selected fromthe group consisting of HCDR1, HCDR2 and HCDR3, wherein said modifiedfirst complementarity-determining region comprises a different aminoacid at one or more positions when compared to the corresponding donorcomplementarity determining regions of said first reference sequence andii) one or more portions of unmodified framework regions which arecapable of hybridizing to said first oligonucleotides; c) mixing saidfirst oligonucleotides with said population of second oligonucleotidesas to create overlapping oligonucleotides; and d) treating saidoverlapping oligonucleotides under conditions such that a population ofaltered heavy chain variable region encoding nucleic acids isconstructed, wherein the framework regions encoded by said altered heavychain variable region encoding nucleic acids are unmodified with respectto said second reference sequence.
 2. The method of claim 1, whereinsaid representation of first and second reference sequences is inelectronic form.
 3. The method of claim 1, further comprising the stepof (E) coexpressing said population of altered heavy chain variableregion encoding nucleic acids with a light chain variable regionencoding nucleic acid so as to produce a diverse population of alteredheteromeric variable regions.
 4. The method of claim 1, wherein saidsynthesizing comprises chemically synthesizing.
 5. The method of claim1, wherein said acceptor is human.
 6. The method of claim 1, whereinsaid treating of step D) comprises extension by a polymerase.
 7. Amethod of constructing a population of altered light chain variableregion encoding nucleic acids, comprising: a) providing a representationof first and second reference amino acid sequences, said first referencesequence comprising the sequence of a donor light chain variable region,said donor variable region comprising i) framework regions and ii) threecomplementarity-determining regions as defined by the combineddefinitions of Kabat and Chothia; said second reference sequencecomprising the sequence of an acceptor light chain variable regioncomprising framework regions; b) synthesizing a) first oligonucleotidesencoding portions of said framework regions of said acceptor light chainvariable region, wherein said portions of said framework regions whencompared to said second reference sequence are unmodified; and b) apopulation of second oligonucleotides, each encoding i) at least aportion of a first complementarity-determining region that has beenmodified, said first complementarity-determining region selected fromthe group consisting of LCDR1, LCDR2 and LCDR3, wherein said modifiedfirst complementarity-determining region comprises a different aminoacid at one or more positions when compared to the corresponding donorcomplementarity determining regions of said first reference sequence andii) one or more portions of unmodified framework regions which arecapable of hybridizing to said first oligonucleotides; c) mixing saidfirst oligonucleotides with said population of second oligonucleotidesas to create overlapping oligonucleotides; and d) treating saidoverlapping oligonucleotides under conditions such that a population ofaltered light chain variable region encoding nucleic acids isconstructed, wherein the framework regions encoded by said altered lightchain variable region encoding nucleic acids are unmodified with respectto said second reference sequence.
 8. The method of claim 7, whereinsaid representation of first and second reference sequences is inelectronic form.
 9. The method of claim 7, further comprising the stepof (E) coexpressing said population of altered light chain variableregion encoding nucleic acids with a heavy chain variable regionencoding nucleic acid so as to produce a diverse population of alteredheteromeric variable regions.
 10. The method of claim 7, wherein saidsynthesizing comprises chemically synthesizing.
 11. The method of claim7, wherein said acceptor is human.
 12. The method of claim 7, whereinsaid treating of step D) comprises extension by a polymerase.
 13. Amethod of constructing a population of altered heavy chain variableregion encoding nucleic acids, comprising: A) providing a representationof first and second reference amino acid sequences, said first referencesequence comprising the sequence of a donor heavy chain variable region,said donor variable region comprising i) framework regions and ii) threecomplementarity-determining regions as defined by the combineddefinitions of Kabat and Chothia; said second reference sequencecomprising the sequence of an acceptor heavy chain variable regioncomprising framework regions; B) synthesizing a) a population of firstoligonucleotides, each encoding at least a portion of a firstcomplementarity-determining region selected from the group consisting ofHCDR1, HCDR2 and HCDR3, wherein said modified firstcomplementarity-determining region comprises a different amino acid atone or more positions when compared to the corresponding donorcomplementarity determining regions of said first reference sequence;and b) second oligonucleotides encoding i) portions of said frameworkregions of said acceptor heavy chain variable region, wherein saidportions of said framework regions when compared to said referencesequence are unmodified and ii) one or more portions of acomplementarity determining region which are capable of hybridizing tosaid population of first oligonucleotides; C) mixing said population offirst oligonucleotides with said second oligonucleotides as to createoverlapping oligonucleotides; and D) treating said overlappingoligonucleotides under conditions such that a population of alteredheavy chain variable region encoding nucleic acids is constructed,wherein the framework regions encoded by said altered heavy chainvariable region encoding nucleic acids are unmodified with respect tosaid second reference sequence.
 14. The method of claim 13, wherein saidrepresentation of first and second reference sequences is in electronicform.
 15. The method of claim 13, further comprising the step of (E)coexpressing said population of altered heavy chain variable regionencoding nucleic acids with a light chain variable region encodingnucleic acid so as to produce a diverse population of alteredheteromeric variable regions.
 16. The method of claim 13, wherein saidsynthesizing comprises chemically synthesizing.
 17. The method of claim13, wherein said acceptor is human.
 18. The method of claim 13, whereinsaid treating of step D) comprises extension by a polymerase.
 19. Amethod of constructing a population of altered light chain variableregion encoding nucleic acids, comprising: A) providing a representationof first and second reference amino acid sequences, said first referencesequence comprising the sequence of a donor light chain variable region,said donor variable region comprising i) framework regions and ii) threecomplementarity-determining regions as defined by the combineddefinitions of Kabat and Chothia; said second reference sequencecomprising the sequence of an acceptor light chain variable regioncomprising framework regions; B) synthesizing a) a population of firstoligonucleotides, each encoding at least a portion of a firstcomplementarity-determining region selected from the group consisting ofLCDR1, LCDR2 and LCDR3, wherein said modified firstcomplementarity-determining region comprises a different amino acid atone or more positions when compared to the corresponding donorcomplementarity determining regions of said first reference sequence;and b) second oligonucleotides encoding i) portions of said frameworkregions of said acceptor light chain variable region, wherein saidportions of said framework regions when compared to said referencesequence are unmodified and ii) one or more portions of acomplementarity determining region which are capable of hybridizing tosaid population of first oligonucleotides; C) mixing said population offirst oligonucleotides with said second oligonucleotides as to createoverlapping oligonucleotides; and D) treating said overlappingoligonucleotides under conditions such that a population of alteredlight chain variable region encoding nucleic acids is constructed,wherein the framework regions encoded by said altered light chainvariable region encoding nucleic acids are unmodified with respect tosaid second reference sequence.
 20. The method of claim 19, wherein saidrepresentation of first and second reference sequences is in electronicform.
 21. The method of claim 19, further comprising the step of (E)coexpressing said population of altered light chain variable regionencoding nucleic acids with a heavy chain variable region encodingnucleic acid so as to produce a diverse population of alteredheteromeric variable regions.
 22. The method of claim 19, wherein saidsynthesizing comprises chemically synthesizing.
 23. The method of claim19, wherein said acceptor is human.
 24. The method of claim 19, whereinsaid treating of step D) comprises extension by a polymerase.